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Monitoring angiogenesis in soft-tissue engineered constructs forcalvarium bone
regeneration: an in-vivolongitudinal DCE-MRI study
Marine Beaumont, Marc G. DuVal, Yasir Loai, Walid A. Farhat, George K. Sándor, Hai-Ling Margaret Cheng
Version Post-print/accepted manuscript
Citation (published version)
Beaumont, Marine, Marc G. DuVal, Yasir Loai, Walid A. Farhat,
George K. Sandor, and Hai‐Ling Margaret Cheng. "Monitoring
angiogenesis in soft‐tissue engineered constructs for calvarium bone
regeneration: an in vivo longitudinal DCE‐MRI study." NMR in
Biomedicine: An International Journal Devoted to the Development and Application of Magnetic Resonance In vivo 23, no. 1 (2010): 48-55.
Publisher’s Statement This is the peer reviewed version of the following article:
Beaumont, Marine, Marc G. DuVal, Yasir Loai, Walid A. Farhat,
George K. Sandor, and Hai‐Ling Margaret Cheng. "Monitoring
angiogenesis in soft‐tissue engineered constructs for calvarium bone
regeneration: an in vivo longitudinal DCE‐MRI study." NMR in
Biomedicine: An International Journal Devoted to the Development and Application of Magnetic Resonance In vivo 23, no. 1 (2010): 48-55. which has been published in final form at 10.1002/nbm.1425. This article may be used for non-commercial purposes in accordance with Wiley Terms and Conditions for Use of Self-Archived Versions.
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Monitoring angiogenesis in soft-tissue engineered constructs for
calvarium bone regeneration: an in-vivo longitudinal DCE-MRI study
Marine Beaumont, PhD1,2
, Marc G. DuVal, MD3, Yasir Loai
4, Walid A. Farhat, MD
1,4, George
K. Sándor, MD3,5,6
, Hai-Ling Margaret Cheng, PhD1,2,7
1The Research Institute, The Hospital for Sick Children, Toronto, Canada
2Diagnostic Imaging, The Hospital for Sick Children, Toronto, Canada
3Department of Oral and Maxillofacial Surgery, University of Toronto, Toronto, Canada
4Division of Urology, The Hospital for Sick Children, Toronto, Canada
5Regea Institute for Regenerative Medicine, University of Tampere, Tampere, Finland
6Department of Oral and Maxillofacial Surgery, University of Oulu, Oulu, Finland
7Department of Medical Biophysics, University of Toronto, Toronto, Canada
Corresponding author:
Hai-Ling Margaret Cheng, Ph.D.
The Hospital for Sick Children
555 University Avenue, Toronto, Ontario M5G 1X8 Canada
Phone: 416-813-5415
Fax: 416-813-7362
Grants: Canadian Institutes of Health Research, Pediatric Regenerative Medicine Fund
Running title: DCE-MRI in tissue-engineered calvarium bone.
DCE-MRI in tissue-engineered calvarium bone 2
ABSTRACT
Tissue engineering is a promising technique for bone repair and can overcome the major
drawbacks of conventional autogenous bone grafting. In this in-vivo longitudinal study, we
proposed a new tissue-engineering paradigm: inserting a biological soft-tissue construct within
the bone defect to enhance angiogenesis for improved bone regeneration. The construct acts as a
resorbable scaffold to support desired angiogenesis and cellular activity and as a vector of
vascular endothelial growth factor, known to promote both vessel and bone growth. Dynamic
contrast-enhanced magnetic resonance imaging was performed to investigate and characterize
angiogenesis necessary for bone formation following the proposed paradigm of inserting a
VEGF-impregnated tissue-engineered construct within the critical-sized calvarial defect in the
membranous parietal bone of the rabbit. Results show that a model-free quantitative approach,
the normalized initial area under the curve metric, provides sensitive and reproducible measures
of vascularity that is consistent with known temporal evolution of angiogenesis during bone
regeneration.
Keywords: Dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI), tissue
engineering, angiogenesis, bone regeneration.
Abbreviations used: DCE-MRI, dynamic contrast-enhanced magnetic resonance imaging, AIF,
arterial input function, IAUC, initial area under the curve, VEGF, vascular endothelial growth
factor, ACM, acellular matrix, HA, hyaluronic acid, IV, intravenous, FA, flip angle, BW,
bandwidth, NEX, number of experiments, 3D, three dimensional, SPGR, spoiled gradient
recalled echo, ROI, region of interest, micro-CT, micro-computed tomography.
DCE-MRI in tissue-engineered calvarium bone 3
GRAPHICAL ABSTRACT
Monitoring angiogenesis in soft-tissue engineered constructs for calvarium bone
regeneration: an in-vivo longitudinal DCE-MRI study
Marine Beaumont, Marc G. DuVal, Yasir Loai, Walid A. Farhat, George K. Sándor, Hai-Ling
Margaret Cheng.
DCE-MRI was performed to
investigate and characterize
angiogenesis necessary for bone
formation following the proposed
paradigm of inserting a VEGF-
impregnated tissue-engineered
construct within the critical-sized
calvarial defect in the rabbit.
Results show that a model-free
quantitative approach, the
normalized initial area under the
curve metric, provides sensitive and
reproducible measures of
vascularity that is consistent with
known temporal evolution of
angiogenesis during bone
regeneration.
DCE-MRI in tissue-engineered calvarium bone 4
INTRODUCTION
Autogenous bone grafting is considered the gold standard for repair and regeneration of
extensive bony defects following trauma, cancer resection, or non-united fractures (1). However,
grafting bone is non-ideal: a second surgical site, usually involving the iliac crest, tibia,
calvarium, or scapula, is required; the bony skeleton does not offer a limitless supply of bone to
harvest; the procedure is associated with increased operating room time usage and increased
patient morbidity (2). Tissue engineering is one of the most promising techniques to overcome
these major drawbacks of autogenous bone grafting (3). Successful tissue regeneration mainly
depends on the establishment of a vascular bed, which connects the implanted construct to the
host tissue and supplies necessary nutrients for bone regeneration (4). The different mechanisms
underlying bone repair and regeneration have been extensively studied and identified (5,6), but
very few studies have focused on monitoring the angiogenesis process during bone wound
healing.
As a noninvasive imaging technique, MRI is well suited to in-vivo longitudinal
evaluation of tissue repair following grafting procedures. In tissue-engineered constructs, MRI
has been used to determine cellularization and maturation of the construct in vivo in cartilage
(7,8), but bony constructs have been studied only in vitro (9,10). In either case, the vascular
status and evolution of angiogenesis with tissue growth were not investigated. Angiogenesis in
tissue-engineered constructs was first evaluated in bladder constructs in vivo (11-13) using
dynamic contrast-enhanced MRI (DCE-MRI). The foray of DCE-MRI into tissue-engineering
applications remains limited despite its well-established role in assessing the microvasculature in
clinical and research studies (14). A few exceptions are found in in-vivo longitudinal studies
conducted using DCE-MRI to evaluate the wound-healing process following subcutaneous
DCE-MRI in tissue-engineered calvarium bone 5
insertion of wound chambers (15) and massive bone allograft (16). In this latter study,
revascularization was assessed at the osteotomy site but not at the graft site. To our knowledge,
longitudinal in-vivo characterization of angiogenesis in bony constructs has never been reported.
In order to quantify angiogenesis using DCE-MRI derived data, one can use numerous
analysis techniques. The quantitative approach using pharmacokinetic modeling of tracer
concentration (17) provides parameters related to physiology, such as blood volume and vascular
permeability. This approach depends on the knowledge of the tracer concentration evolution in
the blood, or arterial input function (AIF). However, determination of the AIF may be
compromised by tracer concentration-MR signal non-linearity, low temporal resolution, and
partial volume effects, which are especially problematic in small animal brain imaging.
Moreover, it has been shown that errors in AIF measurement can strongly influence
pharmacokinetic parameter accuracy (18,19). To overcome this limitation, a model-free
quantitative approach has been proposed (20), which uses the initial area under the tracer
concentration-time curve (IAUC) as an empirical quantitative parameter. Change in the IAUC
has been shown to correlate with vessel density (13) and tumor response to anti-vascular
treatment (21,22). Also, this non-model-based analysis is very robust, and reproducibility is often
comparable to or even better than that obtained with traditional model-based parameters (23-25).
In this in-vivo longitudinal study, we propose a new tissue-engineering paradigm:
inserting a biological soft-tissue construct within the bone defect to enhance angiogenesis for
improved bone regeneration. The construct acts as a resorbable scaffold to support desired
angiogenesis and cellular activity and as a vector of vascular growth factor (VEGF), known to
promote both vessel and bone growth (26). DCE-MRI was performed to investigate and
characterize angiogenesis necessary for bone formation following the proposed paradigm of
DCE-MRI in tissue-engineered calvarium bone 6
inserting a VEGF-impregnated tissue-engineered construct within the critical-sized calvarial
defect in the membranous parietal bone of the rabbit (27). Results show that an IAUC approach
provides sensitive and reproducible measures of vascularity that is consistent with known
temporal evolution of angiogenesis during bone regeneration.
EXPERIMENTAL
This study was approved by the institutional Animal Care Committee (protocol #7578).
Construct Preparation and Implantation
Tissue-engineered construct preparation began with the acellularization of urinary
bladder harvested from 20-50 kg porcines. Bladders were washed with sterile phosphate buffer
saline, longitudinally sectioned and stirred in hypotonic solution for 48 hours at 4°C to
effectively break down cellular structures and inhibit proteases. Bladders were placed in
hypertonic solution for 48 hours at 4°C to denature protein residues and degrade all DNA and
RNA components. Bladders were washed with Hank’s Balanced Salt Solution (Invitrogen,
#14175, USA) containing 2 U/mL Benzonase (Novagen, #: 70746, Germany) overnight at 37°C
and transferred to 0.25% CHAPS-containing detergent based solution. Acellular matrices
(ACMs) were repeatedly washed with sterile dH2O and stored in 70% ethanol prior to use. H&E
staining was used to confirm acellularity. ACMs were cut into 1.5 cm diameter discs, weighed,
and immersed in gradually increasing concentrations of ethanol for full dehydration. ACMs were
lyophilized (ViTis-temp, 120 millitorr and vacuum) for 24 hours, then rehydrated with increasing
concentrations of HA (0.05, 0.1, 0.2 and 0.5 mg/100mL) (Sigma, product #H5388, USA). Alcian
blue staining was used to confirm HA incorporation. In addition, HA-ACMs were dehydrated in
alcohol, lyophilized, and rehydrated with VEGF121 (Sigma, #V3388, USA) 10 ng/g of ACM.
DCE-MRI in tissue-engineered calvarium bone 7
Finally, a critical-sized (non-spontaneous healing) calvarial defect (15 mm in diameter) was
created surgically in the parietal bones of the rabbits, the centre of the defect was 9.5 mm from
the midline to allow 2 mm for the sagittal sinus. The tissue-engineered HA-VEGF containing
construct was grafted to the calvarium.
Experimental Protocol
Surgery and MR imaging were performed on twelve New Zealand white rabbits (3.5-
4 kg). Imaging sessions were held 1, 2, 3, 6 and 12 weeks after the surgery. All MR experiments
were performed under anesthesia using two different protocols. Five rabbits were anesthetized
with an intravenous (IV) injection of pentobarbital (25 mg/kg) through the ear vein. During the
MRI (about 1 hour), anesthesia was maintained with an IV dose of 0.25 mg/kg/min; and the
animals were kept under 100% oxygen with a face mask. For the other seven rabbits, anesthesia
was performed with 5% of isoflurane for induction, and 2±0.2% for maintenance, in 100%
oxygen using a face mask. All the animals were equipped with a catheter in the ear vein for the
contrast agent injection (Gd-DTPA – Magnevist, Berlex Canada, Lachine, Canada) and
continuous hydration (4 mL of saline/kg/h). Heart rate and pO2 were monitored during the scan
time. Five animals were euthanized after the fourth imaging session (6 weeks after surgery)
while the remaining animals were euthanized after the last imaging session (12 weeks after
surgery). In each animal, the calvarium was excised for subsequent histological procedures.
MRI
MRI was performed on a 1.5 T GE scanner (Signa EXCITE TwinSpeed; GE Healthcare,
Milwaukee, WI, USA) using a three-inch diameter receive surface coil and a body transmit coil.
Animals were placed in prone position and an axial orientation was chosen for all the slices.
Construct localization was achieved using a gradient echo sequence: TR/TE = 6.5/2.3 ms,
DCE-MRI in tissue-engineered calvarium bone 8
FA = 30°, bandwidth (BW) = 23.4 kHz, 16 x 12 x 8 slices (thickness = 2 mm, space between
slices = 1 mm), matrix = 192 x 192, FOV = 12 x 12 cm2, number of experiments (NEX) = 2. A
rapid 3D T1-mapping method, based on variable flip angles and integrating B1 correction (28),
was employed prior to Gd-DTPA injection to acquire the baseline T1 map: 3D fast spoiled
gradient recalled echo (SPGR), TR/TE = 8.8/3.4 ms, FA = 2°, 10° and 20°, bandwidth
(BW) = 31.2 kHz, matrix = 256 x 160 x 10, FOV = 10 x 10 x 3 cm3, number of experiments
(NEX) = 4. Dynamic T1-weighted images were acquired using a 3D fast SPGR sequence:
TR/TE = 10.2/3.4 ms, FA = 60°, BW = 31.2 kHz, matrix = 256 x 128 x 10,
FOV = 10 x 10 x 3 cm3, NEX = 0.75. After the acquisition of 5 baseline images, Gd-DTPA was
administered as a rapid bolus (0.1 mmol of Gd/kg) and imaging continued for 6 min post-
injection with a time resolution of 8.2 s (300 images in total).
MRI data analysis
All analyses were performed on a pixel-wise basis using in-house programs developed
with Matlab (v.7.0, Mathworks Inc., Natick, MA, USA). Analysis of the 3D T1 maps followed
the method described previously (28). DCE-MRI data were converted into Gd concentration
maps using the pre-injection T1 map, the SPGR signal equation (29) and the Gd-DTPA relaxivity
r1 (4.1 s-1
.mM-1
, in plasma at 1.5 T and 37°C (30)). Finally, the initial area under the Gd
concentration time curve for 60s after contrast agent injection (IAUC60) was computed for each
pixel. Only physiologically meaningful pixels were retained. Pixels were excluded if they met
any of the following conditions: T1 values outside of the range 0-4000 ms, Gd concentration
mean value over time outside of the range 0-2 mM and for which the standard deviation of Gd
concentration along the baseline was greater than 0.1 mM. Three regions of interest (ROI) were
manually drawn. On the IAUC60 map, two ROIs were defined on the construct: the periphery,
DCE-MRI in tissue-engineered calvarium bone 9
which corresponds to the early enhancing region, and the centre (Figure 1c); and one ROI
surrounding the whole brain. For each ROI and for each MR acquisition time point, the mean
IAUC60 value was computed as well as the percentage of physiologically meaningful pixels in
the ROI involved in this calculation. To minimize effects from inter-individual physiological
variation and contrast agent dose variability, each IAUC60 mean value was normalized to a
reference, the IAUC60 mean value measured in the brain.
Statistical analysis was based on the permutation test (also called randomization test,
(31)), which, unlike the widely used t-test, is suited to data originating from small-sized groups
and with unknown distributions. This test determines the significance of the difference of means
d between two populations A and B with nA and nB samples, respectively, as follows. A
difference of means d’ is calculated between all possible groupings A’ and B’, where A’ and B’
represent a different partition of values from A+B into two groups of size nA and nB. The P-value
P is equal to the ratio of the number of events where d’>d to the total number of events. Paired
permutation tests were applied to compare IAUC60 at different time points, and statistical
significance was set at P < 0.05. Evolution of the IAUC60 mean value with time was also
determined for each individual animal and in each ROI by comparing the mean value of the
estimate at week (i) with the mean±SEM value at week (i-1).
Micro-Computed Tomography
Calvarial specimens were scanned by an Explore Locus SP micro-CT scanner (GE
Medical Systems, London, Ontario, Canada) using the fast mode with 0.05 mm sections.
Reconstruction of scanned images was done using Microview software (GE Medical Systems,
London, Ontario, Canada) after calibrating using the bone water and air standard values. The
reconstructed 3D image was then traced in 3 dimensions to the circumference of the original
DCE-MRI in tissue-engineered calvarium bone 10
defect margins. This allowed the creation of a 3D reconstruction of the defect, referred to as the
ROI. A threshold level was selected manually based on 25% of the bone standard provided by
the manufacturer.
RESULTS
MRI
Figure 1 illustrates that the soft-tissue construct was clearly identifiable on MR images
even prior to contrast agent injection (Fig.1a). The T1-weighted MR signal increased in the
whole construct after contrast agent injection with a more pronounced enhancement in the
periphery than in the centre of the graft (Fig.1b). Note that contrast uptake was observed from
the first imaging time point (1 week after surgery) up to the last (12 weeks after surgery) in all
rabbits. The IAUC60 map provided the best support to draw the different ROIs with very clean
contours of the construct (Fig.1c).
Table 1 shows the evolution of the percentage of physiologically meaningful pixels in
these ROIs that are retained for DCE-MRI analysis. The percentage of pixels retained is
generally highest ( 91%) in the construct periphery compared to the centre or normal brain
tissue. An exception occurs at 12 weeks, where fewer useful pixels are detected in the entire
construct. In the centre, the percentage of retained pixels increases with angiogenic growth. In
the normal brain where contrast agent uptake is lowest, at least 75% of pixels were retained
consistently throughout the 12 weeks, which supports the robustness of the proposed method for
pixel selection. Table 2 reports the evolution of the size of the ROIs normalized to week 1. For
both periphery and centre of the construct, the size of the ROI 6 weeks after the surgery is almost
half of the initial size. The size of the ROI defined on the brain remains constant with time.
DCE-MRI in tissue-engineered calvarium bone 11
Figure 2 illustrates in one rabbit typical uptake curves in the periphery and the centre of
the construct at four time points (1, 3, 6 and 12 weeks after surgery). The uptake curves in the
periphery were characteristic of highly permeable vessels usually found in presence of active
angiogenesis. At the early time point, the periphery enhancement curve (Fig.2, Week 1) exhibits
the most rapid initial uptake followed by a distinct washout. Later time points may also show
significant enhancement, but the initial uptake is more gradual and washout is not always evident
(e.g. Fig.2, Week 6). In the centre of the construct, the uptake dynamics were distinct from those
in the periphery, characterized by a much lower and gradual uptake. Both ROIs exhibited less Gd
uptake at the last time point of 12 weeks after graft implantation.
Figure 3 shows the evolution of the mean IAUC60 values normalized to the reference
(IAUC60 in the brain) in both the periphery and centre of the construct throughout the 12 weeks.
The periphery exhibited the highest value at the early time point, followed by a plateau and a
two-fold decrease at 12 weeks post-surgery. The centre of the construct exhibited different
characteristics. Normalized IAUC60 values were significantly lower at all time points, and a
progressive increase was observed at early times up to 3 weeks post-surgery. The one common
trait observed in both the centre and periphery was a decrease in uptake at 12 weeks. Values for
all animals are shown in Table 3.
Figure 4 shows how angiogenesis evolves in individual animals at time points where
significant changes were observed, namely, between weeks 1 and 3 and weeks 6 and 12. It also
distinguishes groups administered different anesthetics (solid versus dashed lines). Several key
results are noted in this figure. First, the proposed DCE-MRI approach yields reproducible trends
in angiogenic development in most animals: a significant early decrease in the periphery versus
an increase in the centre (in each case, the trend was observed on 8 animals out of 12), followed
DCE-MRI in tissue-engineered calvarium bone 12
by a decrease in the entire construct at 12 weeks. Second, this approach is insensitive to
differential vascular response to anesthesia that is independent of angiogenesis. An exception to
this is seen at 12 weeks (Table 3), where normalized IAUC60 values are significantly higher in
the isoflurane group. Although the sample size was small (n=2), the possibility of anesthesia
influence on vascular development at 12 weeks could not be disregarded, and these two animals
were excluded from the twelve weeks mean values in Fig. 3 and Table 3. It is important to note
that aside from the 12-week discrepancy, these two animals followed the general trend at earlier
time points and were part of the 8 out of 12 animals showing the same behavior between week 1
and week 3. Further investigations are required to confirm this hypothesis regarding anesthesia-
induced differences.
Micro-Computed Tomography
Figure 5 shows representative micro-CT images of excised calvarium. Micro-CT
evaluation supported the MRI findings. There was new bone noted at 6 weeks, mostly at the
periphery of the bony defects containing the HA-VEGF constructs, while at 12 weeks islands of
newly formed bone had appeared in the center of the defects.
DISCUSSION
Tissue engineering is a promising technique for bone repair and can overcome the major
drawbacks of conventional autogenous bone grafting. Instead of using a surgically harvested
bone graft, a biological soft-tissue construct is placed within the bony defect. This construct acts
as a resorbable scaffold to support two intimately linked processes during tissue regeneration:
cellular colonization and angiogenesis. Monitoring angiogenesis longitudinally, for instance,
using DCE-MRI, provides a means to assess bone development non-invasively. In this study, a
DCE-MRI in tissue-engineered calvarium bone 13
model-free analysis of DCE-MRI data was used to investigate and characterize angiogenesis in a
VEGF-impregnated tissue-engineered construct within the rabbit calvarial defect. Vessel
network initiation and establishment within the construct, leading to successful bone
regeneration, were demonstrated in vivo using normalized IAUC60 measurements. The proposed
approach provides reproducible and sensitive results and is robust to challenging experimental
conditions.
Natural wound healing and bone regeneration after autogeneous bone grafting have been
extensively described in the literature. Bone regeneration results from a combination of different
processes, or phases, which occur usually in a dynamic equilibrium (6). Although our proposed
paradigm uses a soft-tissue construct instead of a bone graft, the same phases are required to
ensure successful bone regeneration. In fact, the collagen-based scaffold may be more easily
resorbed than traditional bone grafts to allow new bone formation. Also, the soft-tissue construct
provides the ideal three-dimensional framework to guide desired angiogenesis (4), which is a
major determinant to bone formation. The different processes involved in bone formation are as
follows. The early inflammatory phase is characterized by the different components of wound
healing: inflammation, hemorrhage, hematoma, and blood clot formation. These combined
phenomena stimulate macrophages to release growth factors (such as VEGF), which induces
angiogenesis (32). Vascularization is the second stage in graft healing and is strongly associated
with the previous phase. It starts shortly after graft implantation, and the mature vessel network
is established within 2 to 3 weeks following surgery. The third phase significantly overlaps with
the vascularization phase and may last up to 6 weeks. During this time span, the graft is
colonized by cells, nutrients, and other growth factors necessary for new bone formation via the
newly established vasculature. The final phase, the remodeling phase, is initiated between the
DCE-MRI in tissue-engineered calvarium bone 14
fourth and the sixth week post-surgery and continues for several months. It involves remodeling
and mineralization of the immature randomly-oriented bone. The two latter phases are also
accompanied by the resorption of the graft material (6).
In our study, periphery and centre regions of the soft-tissue construct exhibited different
behaviors with regards to angiogenic activity, but in each case, the different phases observed
were consistent with expected angiogenic progression, as described in the literature. In the
periphery, the high IAUC60 value observed one week after the surgery is consistent with the first
two phases of inflammation and angiogenesis, since both lead to high contrast agent uptake due
to high vessel leakiness. Then, between the second and the sixth week after insertion, when both
angiogenesis and the third phase are expected to occur, the IAUC60 is lower and remains
relatively constant. The lower contrast agent uptake is consistent with the completion of the
inflammatory phase. The plateau in IAUC60 needs to be interpreted carefully, since different
uptake curve dynamics (at weeks 3 and 6) suggest distinct vascular behavior that possibly
indicates a transition from phase 2 to 3. The IAUC60 contribution comes mainly from
inflammatory/angiogenic activity initially and later from a mature vasculature that is functioning
fully to supply the graft with elements to the new bony tissue. Finally, the drop at twelve weeks
is expected since the remodeling phase no longer requires a large vascular supply, perhaps
resulting in subsequent vessel pruning (33). In the centre, the inflammation phase is not
distinguishable because of the distance from the actual surgery site. As for the three other phases,
they are clearly separated: the slow IAUC60 increase reflects the growing vessels during
angiogenesis, the plateau corresponds to the new bone formation phase, and the decrease is the
same as in the periphery. Nevertheless, it should be pointed out that the uptake curve pattern
observed in the centre may reflect primarily diffusion of the contrast agent from the construct
DCE-MRI in tissue-engineered calvarium bone 15
periphery to the core and is not a purely vascular-driven response. In fact, diffusion is a
fundamental means of transport in tissue regeneration since some elements, like cells, are not
vehicle from the host tissue onto the graft via the blood, but using diffusion along the scaffold.
Finally, the size of ROIs defined on the construct decreased significantly from week 1, indicating
resorption of the graft while new bony tissue is created as observed on the micro-CT results.
In this work, the IAUC approach was preferred to more common model-based
approaches to analyze DCE-MRI data. Indeed, our experimental conditions (small animal brain
imaging with clinical magnetic field, low temporal resolution) rendered the determination of the
AIF very challenging and would have compromised a model-based analysis (19). Although
empirical approaches do not have clear physiological associations, our results show that our
normalized IAUC60 metric is very sensitive to the different stages that occur during graft healing
and bone regeneration, and can monitor the regeneration process and its success. Moreover,
when quantitative model-based approaches are used, numerous factors (AIF errors, limited
temporal resolution, choice of analysis model) may impair robustness and accuracy (18). When
using an IAUC metric, several approaches have been taken. Walker-Samuel et al. (34)
recommended the use of IAUC90 for in-vivo studies. But they also mentioned that the integration
period does not matter if the interval is long enough to ensure good signal-to-noise ratio. In
another study, Morgan et al. (24) showed there were no major changes by using IAUC60 or
IAUC180. Furthermore, they demonstrated that reproducibility was improved when IAUC was
computed from the T1 values rather than from the MR signal intensities. Finally, Evelhoch et al.
(21) proposed normalizing the data with an AIF for longitudinal studies. Considering all these
elements, we used normalized IAUC60 values derived from the Gd concentration time curve to
achieve accuracy. Although our normalization was performed using the whole brain IAUC60
DCE-MRI in tissue-engineered calvarium bone 16
value rather than the AIF one, the automatic selection of enhancing pixels implemented provided
results sensitive enough to minimize effects from inter-individual physiological variation and
contrast agent dose variability. Muscle tissue was not chosen as a reference in this study because
of low signal-to-noise ratio due to greater distance from the surface coil. The good sensitivity of
the IAUC approach demonstrated in this study warrants further investigation to achieve
improved distinction of uptake curve patterns.
The presented normalized IAUC60 method also demonstrated robustness and insensitivity
to the anesthetic procedure (one exception occured at the last time point), whereas the non-
normalized IAUC60 values presented significant differences (results not shown). Indeed, the two
anesthetics used in our protocol are well known to induce antagonistic physiological effects,
mainly on the vasculature (35). Those effects have been investigated using MRI and they have
been shown to influence the MR signal (36). As demonstrated by Hendrich et al. (37), using
arterial spin labeling on rat, cerebral blood flow, as well as pO2 and mean arterial blood pressure,
measured on animals undergoing isoflurane anesthesia are higher than the ones of animals
anesthetized with pentobarbital. If the proposed approach successfully homogenized the two
groups without hiding the relevant changes in angiogenesis activity, a discrepancy appeared at 12
weeks after surgery. Several hypotheses may be raised to explain this result. First, one can
suggest that the sample size of the isoflurane group at week 12 (n = 2), is not large enough to
reflect an actual difference, and this result may be biased by inter-animal variability. Another
hypothesis, as mentioned above, is long-term remodeling of the newly established vasculature,
which normally leads to partial vessel pruning. The repeated isoflurane anesthesia could have an
anti-pruning effect on the vasculature, resulting in a higher blood volume and, thus, a higher
normalized IAUC60 value. This explanation is likely, since isoflurane is known for having
DCE-MRI in tissue-engineered calvarium bone 17
preconditioning properties, as shown in stroke (38). Finally, as isoflurane and pentobarbital
generate vasodilatation and vasoconstriction, respectively, the trend observed may be simply an
emphasis of the natural trend, because vessels are more mature and more reactive to those
chemical stimulations. A combination of these three hypotheses may also explain the reported
difference. Understanding this phenomenon requires further investigation.
In conclusion, this study provides the first report of an in vivo longitudinal DCE-MRI
study in tissue-engineered calvarium bone. The soft-tissue construct acts as a resorbable support
for promoting angiogenesis and achieving complete bone regeneration. The normalized IAUC60
approach used for the DCE-MRI analysis was capable of characterizing angiogenesis and
discriminating between the different phases of bone regeneration, and was robust to challenging
experimental conditions.
ACKNOWLEDGMENTS
We thank Marvin Estrada for animal care support and Tammy Rayner, Ruth Weiss, and
Garry Detzler for technical assistance during MR scanning.
DCE-MRI in tissue-engineered calvarium bone 18
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DCE-MRI in tissue-engineered calvarium bone 22
Table 1: Evolution of the percentage of physiologically meaningful pixels (within the ranges of
validity defined in MRI data analysis).
Physiologically meaningful pixels (% - mean ± SEM)
Time after surgery (weeks)
ROI n 1 2 3 6 12 n
Periphery 12 94 ± 2 94 ± 3 92 ± 1 91 ± 2 75 ± 6 5
Centre 12 77 ± 4 80 ± 5 93 ± 3 91 ± 4 69 ± 6 5
Brain 12 75 ± 2 75 ± 2 71 ± 3 78 ± 2 77 ± 3 5
DCE-MRI in tissue-engineered calvarium bone 23
Table 2: Evolution of the size of the ROI normalized to week 1.
Size of the ROI normalized to week 1 (% - mean ± SEM)
Time after surgery (weeks)
ROI n 1 2 3 6 12 n
Periphery 12 100 88 ± 6 * 88 ± 6 57 ± 6 * 70 ± 12 5
Centre 12 100 73 ± 9 * 72 ± 10 56 ± 7 * 34 ± 6 5
Brain 12 100 101 ± 1 103 ± 2 100 ± 2 102 ± 2 5
* P < 0.05 week (i) versus week (i-1)
DCE-MRI in tissue-engineered calvarium bone 24
Table 3: Evolution of normalized IAUC60 values presented in all animals and in each anesthetic
group.
IAUC60ROI
/ IAUC60brain
(mean ± SEM)
Time after surgery (weeks)
ROI n 1 2 3 6 12 n
Periphery
All 12 8.42 ± 0.48 6.44 ± 0.47 6.56 ± 0.39 6.40 ± 0.57 3.44 ± 0.45 5
Pentobarbital 5 7.93 ± 0.83 5.14 ± 0.41 * 6.73 ± 0.70 5.41 ± 0.44 3.44 ± 0.45 * 5
Isoflurane 7 8.78 ± 0.60 7.37 ± 0.53 * 6.44 ± 0.50 7.10 ± 0.86 7.46 ± 0.69 * 2
Centre
All 12 1.92 ± 0.29 2.15 ± 0.26 2.44 ± 0.22 2.41 ± 0.34 1.35 ± 0.22 5
Pentobarbital 5 1.49 ± 0.13 1.64 ± 0.13 2.13 ± 0.37 2.19 ± 0.73 1.35 ± 0.22 * 5
Isoflurane 7 2.23 ± 0.47 2.51 ± 0.39 2.65 ± 0.26 2.57 ± 0.32 2.95 ± 0.61 * 2
* P < 0.05 Pentobarbital versus Isoflurane
DCE-MRI in tissue-engineered calvarium bone 25
FIGURES LEGENDS
Figure 1: T1-weighted images (a) before, (b) 60s after Gd-DTPA injection and (c) the
corresponding IAUC60 map. Imaging time is 3 weeks post-surgery. A zoomed-in version of the
implanted VEGF-impregnated soft-tissue construct is shown in insets. Note the two distinct
regions of the construct (periphery and centre) on images (b) and (c). The periphery ROI is
delineated on the inset of (c).
Figure 2: Representative Gd-DTPA concentration-time curves in one animal between 1 and 12
weeks post-surgery. Mean values in the periphery and the centre of the implanted construct are
shown.
Figure 3: Evolution of angiogenesis in the periphery and centre of the implanted construct in all
animals (n=12, n=5 for 12 weeks) using the normalized IAUC60 metric. Results are presented as
mean±SEM. P<0.05: different relative to week 1, # different relative to week 6.
Figure 4: Time points showing significant changes in angiogenic development in all animals.
Evolution of the vascular response does not differ between the two anesthetic procedures
(isoflurane (dashed lines) and pentobarbital (solid lines)). On each graph, the number of rabbits
showing an increased (↑), decreased (↓), or constant (=) change is indicated.
Figure 5: Evaluation of bone growth with micro-CT images. (a) At 6 weeks after the surgery,
new bone is present mostly at the periphery of the bony defect containing the HA-VEGF
construct. (b) At 12 weeks, there were islands of newly formed bone in the center of the defect.
The dotted line indicates the midline and the circle is an estimate of the original defect.
Figure 1
Figure 2
b a c
Week 1
0
0.05
0.1
0.15
0.2
0.25
0.3
0.35
0 1 2 3 4 5 6
Time (min)
[Gd
] (m
M)
Periphery
CentreWeek 3
0
0.05
0.1
0.15
0.2
0.25
0.3
0.35
0 1 2 3 4 5 6
Time (min)
[Gd] (m
M)
Periphery
Centre
Week 6
0
0.05
0.1
0.15
0.2
0.25
0.3
0.35
0 1 2 3 4 5 6
Time (min)
[Gd] (m
M)
Periphery
CentreWeek 12
0
0.05
0.1
0.15
0.2
0.25
0.3
0.35
0 1 2 3 4 5 6
Time (min)
[Gd] (m
M)
Periphery
Centre
1
2
3
4
5
6
7
8
9
0 2 4 6 8 10 12
Time after surgery (weeks)
IAU
C60R
OI / IA
UC
60b
rain
Periphery
Centre
Figure 3
#
#
Figure 4
0
1
2
3
4
5
6 12
Time after surgery (weeks)
IAU
C6
0R
OI /
IA
UC
60
bra
in
0
1
2
3
4
5
1 3
Time after surgery (weeks)
IAU
C6
0R
OI /
IA
UC
60
bra
in
0
0.5
1
1.5
2
2.5
3
3.5
4
4.5
5
6 12
Time after surgery (week)
IAU
C60R
OI /
IAU
C60b
rain
(a.u
.) Pentobarbital
Isoflurane
2
4
6
8
10
12
1 3
Time after surgery (weeks)
IAU
C6
0R
OI /
IA
UC
60
bra
in
0
2
4
6
8
10
6 12
Time after surgery (weeks)
IAU
C6
0R
OI /
IA
UC
60
bra
in
Centre Periphery a
c
b
d
↑ 2 = 2 ↓ 8
↑ 8 = 1 ↓ 3
↑ 1 = 2 ↓ 4
↑ 0 = 1 ↓ 6
Figure 5
a b